Secretion of the proteins into the culture medium involves transfer of the proteins through the various membrane-enclosed compartments constituting the secretory pathway. First the proteins are translocated into the lumen of the endoplasmic reticulum, ER. From there on the proteins are transported in membrane vesicles to the Golgi complex and from Golgi to plasma membrane. The secretory process involves several steps in which vesicles containing the secreted proteins are pinched off from the donor membrane, targeted to and fused with the acceptor membrane. At each of these steps functions of several different proteins are needed.
Several attempts have been made to increase protein secretion in filamentous fungi. A common approach to increase secretion of heterologous proteins is to use signal sequences (see for example EP 0215 594). Random mutagenesis and screening for a secreted protein (Smith et al., 1985; Sakai et al., 1988; Shuster et al., 1989; Suzuki et al., 1989; Sleep et al., 1991; Lamsa and Bloebaum, 1990; Dunn-Coleman et al., 1991 and US 2002/0068325 A1) or fusion of the foreign protein to an efficiently secreted endogenous protein (Ward et al., 1990; Harkki et al., 1989; Nyyssonen et al 1993; Nyyssönen et al., 1992) have been widely used both for yeast and filamentous fungi in order to make the secretion of heterologous proteins more efficient. Both of these methods are of limited use. Mutants isolated by random mutagenesis and screening are almost exclusively recessive and thus cannot be transferred into industrial strains, which are polyploids. Often the mutants obtained have only improved secretion capacities for the protein used for screening. Fusion protein approach requires tailoring of the fusion construction for each heterologous or foreign protein separately. The fusion protein is often not functional and thus the final product must be released by proteolytic cleavage, which complicates the production procedure.
Due to their industrial importance as protein producer, there is still a need to obtain filamentous fungi with improved secretion capacity.